General Introduction to Problem Area
Somatic embryogenesis in plants is a process in which somatic embryos are formed from an initial explant being a cell in a plant tissue. The somatic embryos formed are genetically identical copies of the plant providing the initial explant. The process of somatic embryogenesis thereby offers a tool to obtain large numbers of genotypically identical plants for multiplication of selected genotypes of commercial interest, for conservation of endangered species or for generating genetically uniform plant material for research purposes.
Physiological Background to the Procedures Related to the Problem
To produce plants from somatic embryos of conifers, a multi-step procedure is applied to meet the physiological needs of the different stages of development as described below and shown in FIG. 1. Initiation of somatic embryogenesis starts with induction of somatic embryos from an initial explant, typically an immature zygotic embryo, on a solidified culture medium containing plant growth regulator. Somatic embryos continue to form, typically on the same composition culture medium, and a proliferating embryogenic culture form. At the proliferating stage, several of the key features generally regarded as beneficial for the process of somatic embryogenesis process, take place: (i) the mass propagation of genotypically identical propagules through unlimited multiplication of immature embryogenic tissue; (ii) cryogenic storage of proliferating embryos substantiates an virtually eternal store of clones, i.e. a clone bank is established, (iii) transgenic modification of the immature somatic embryo allow for large scale propagation of genetically improved propagules. At the next step in the procedure, the proliferating somatic embryo is subjected to a growth medium that triggers embryo development to progress into the maturation stage. Conversion from proliferation to maturation only occurs in a fraction of the proliferating embryos in the culture. Low conversion rates are encountered more frequently in genotypes from recalcitrant conifer species, but are common in all conifer species as well as other plant species. The manual labour needed to collect embryos increase with the decrease in conversion rate, and thereby the cost and risk of contamination and other inaccuracies. Low conversion rate from proliferation to maturation is a major bottleneck for commercial large scale applications of somatic embryogenesis procedures. For germination, mature somatic embryos are subjected to different culture regimes to induce root- and shoot formation, in a number of different steps; desiccation, sucrose treatment, red light induction, and blue light stimulation. Thereafter, germinated embryos deemed appropriately developed are transferred to a compost material and gradually transferred to an environment ex vitro during which the sucrose content is reduced. The different treatments during germination into a plant requires repeated manual handling of individual germinants and plants adding a considerable cost to the overall procedure.
Production of Plants from Somatic Embryos
The prior art procedure for producing plants from somatic embryos requires manual handling at several steps making the procedure time consuming, expensive and inaccurate.
For conifer species, standard procedures used involve several steps when manual handling is required. The general procedure is outlined in FIG. 1 (see e.g. von Arnold S, Clapham D. Spruce embryogenesis. 2008. Methods Mol Biol. 2008; 427:31-47; Belmonte M F, Donald G, Reid D M, Yeung E C and Stasolla C. 2005. Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca). J Exp Bot, Vol. 56, No. 419, pp. 2355-2364).
There are four steps that rely on manual handling to obtain a small plant from the mature somatic embryo as seen in FIG. 1. The first manual interaction is when [1] the mature embryo is isolated from immature embryos (120), and placed horizontally in a plastic container under sterile conditions; the second [2] occur after 3-7 days of resting (130), then mature embryo is transferred to a gelled culture medium for initiation of germination processes. The germinated somatic embryo will under appropriate culture medium composition and light conditions initiate roots (140). The third manual transfer [3] is when the germinant having a small root formed is transferred to an upright position with the root partially immersed in liquid germination media (150). The fourth [4] and final transfer is when the germinated embryos has a tap root and small lateral roots, then it is transferred into a solid substrate in a pot for further plant formation (160).
TABLE 1List of designations pertaining to FIG. 1.ItemDesignation100Mature embryo101Crown of a mature embryo102Foot of a mature embryo103Width of crown of a mature embryo104Length of a mature embryo120Maturation phase130Resting phase140Germination phase150In vitro plant formation phase160Ex vitro plant formation phase
In the hitherto available method for producing plants from somatic embryos the embryos are picked out manually from the immature embryogenic tissue. This is time-consuming and ineffective. Prior art U.S. Pat. No. 6,684,564 and U.S. Pat. No. 7,568,309 teach automation of the manual transfers by replacing human eye, human arm and tweezers with vision systems, conveyer belts and automated robotic arms with suction tips to pick up the embryos from porous conveyer belts to deposit in a destination much like a human does the same. However, this approach, analogous to automating the wing motion of a bird by robotic wings for flight, is too complex and impractical for several reasons, as elaborated below. It would therefore be desirable to provide a simple and practical way to make the separation and deposition of the embryos faster and more efficient. The mature somatic embryos produced are initially glued together with immature embryogenic tissue in clusters. This makes the process more complex, as embryos have to first be separated from the immature embryogenic tissue in the cluster by breaking up the cluster. Prior art does not teach how to breakup the embryogenic clusters in an automated manner.
Breaking Up the Clusters by a Disperser
In the patent application PCT/US09/39981 a method of rapidly breaking up the clusters is disclosed based on suspending the said clusters in liquid medium, such as water, and forcing the clusters into at least one dispersion sequence where the clusters of embryogenic mass are exposed to flow-dynamic forces causing the breakup of the clusters and dispersion of individual embryos.
Segregation of Embryos by a Separator
When embryogenic mass is dispersed in liquid according to the above-presented method, a mixture of immature and mature embryos and immature embryogenic tissue are suspended in the liquid medium. In many applications, it is highly desirable to segregate and collect the embryos from the dispersed embryogenic mass prior to processing further downstream. For example, if the intention is to image and analyze the shape and condition of the embryos, such as in Harrell et al., 1993 (Computers and Electronic in Agriculture, 9), it is highly desirable to only have embryos suspended in liquid without any of the immature embryogenic tissue in order to avoid obscuring the image. The tasks of image recognition and analysis become more difficult and tedious if the image contains more objects than just the embryo. Furthermore, the task of image processing will be more time consuming with adverse impact on the processing and the conversion rates. Thus there is a need for methods and means for an effective separator to segregate and selectively remove and guide only the embryos from the dispersed embryogenic mass into a separate flow stream in a rapid and efficient manner. Having only individual embryos in a flow stream would facilitate further processing of the embryos which may include digital imaging of the individual embryos, image analysis and characterization of the embryos including identification and control of embryo orientation prior to deposition into an appropriate substrate for germination and plant production.
It is an object of the invention to provide an automated means for gently segregating and separating dispersed somatic embryos from the immature embryogenic tissue and guiding the collected embryos into a separate stream of liquid in a rapid and efficient manner.
Embryo Deposition Means
The prior art methods to make plants from somatic embryos require intensive manual handling, and are therefore expensive for plant production. Attempts to automate the steps used in the manual operation have failed due to the complex devices developed to automate the manual transfer and delivery of embryos by means of moving parts such as conveyer belts and elaborate robotic arms. For example, the prior art documents U.S. Pat. No. 7,568,309 and U.S. Pat. No. 6,684,564 teach means of transferring the embryos into an artificial seed by means of a porous conveyer belt and moving robotic arms equipped with suction tips to pick up the embryo from the conveyer belt and to deposit the embryo by means of a movable robotic arm attached to a rail into an artificial seed. Such processes require many moving parts such as pulleys and motors to drive the conveyer belt, suction device(s) to vacuum excess liquid from the embryo, and elaborate robotic arm assembly movably attached to a rail with precision control to locate and pick the embryo from the conveyer belt. The embryo being a small and delicate object, the robotic arm must have sensitive and precise means of picking and carrying the embryos without damaging it. As explained in U.S. Pat. No. 6,684,564, the conveyer belt must stop moving when an embryo is detected in order for the embryo to be imaged and picked up by mechanical means of a robotic arm. A conveyer belt that has to move and stop each time an embryo is detected creates an inherently inefficient process. In general, the prior art teaches an approach requiring many moving parts including the conveyer belts and the robotic arm assemblies making the current state of the art to be impractical.
Thus, one object of the invention is to provide an advantageous method and device for delivering an embryo to a desired embryo receiver, not requiring any pulleys, conveyer belts, robotic arms or such devices with moving parts.
System
It is another object of the invention to provide a system for processing plant somatic embryos performing the separation process and at least one additional process step of the entire process from a bioreactor to a planted propagule, providing cost-effective means for handling and large-scale production of plants from somatic embryogenesis.
Definitions
For purposes herein, the terms somatic embryo, embryo and plant somatic embryo are used interchangeably. The terms refer to plant embryos derived from somatic tissue of a plant, whether mature or immature.
The term embryogenic mass refers collectively to the plant material consisting of immature embryogenic tissue, or mature embryos and immature embryogenic tissue, present in the liquid or solid culture of somatic embryos.
The term immature embryogenic tissue refers to all material other than embryos that are in the embryogenic mass. The term tissue is being used here in an unconventional manner consisting of largely undifferentiated cells and should not to be confused with the normal reference to plant tissue with specialized cells.
The terms embryogenic clusters, embryo clusters or clusters, are used interchangeably. The term refers to assemblies of plant embryogenic mass held together as a continuous solid material of finite size on solid medium or in liquid medium.
Norway spruce is a spruce species with the Latin name Picea abies native to Europe.
The orthogonal directions in polar coordinates are given by axial (z), radial (r) and angular (or azimuthal) (θ) directions. These directions correspond to the central axis of a cylinder which is normal to the circular cross-sectional of the cylinder. The radial and angular directions point along the radius and normal to the radius on the cross-sectional surface respectively.
Axisymmetric flow refers to flow inside a tube where the cross-sectional surface of the tube is always circular, and therefore, there is symmetry with respect to the axis of the tube. In other words, nothing changes along the angular (or azimuthal) direction.
Pressure gradient refers to the rate of variation of pressure with respect to a given axial direction.
Axial, radial, angular pressure gradient refers to variations in pressure (p) in the axial, radial, and angular directions shown respectively in mathematical terms as partial derivatives
            ∂      p              ∂      z        ,            ∂      p              ∂      r        ,                    ∂        p                    ∂        θ              .  
The term Vortex (plural Vortices), as used here, is a term referred to a flow that possesses vorticity with a spinning or swirling motion around a central axis.
Vortex flow can be categorized as free (irrotational) vortex or forced (rotational) vortex.
As used here, the term Vorticity in mathematical terms, is the curl of the velocity vector field; therefore, it is a vector quantity with magnitude and direction. In other terms, the value of vorticity at a point in the flow is related to rate of rotation of the fluid particles at a point in the flow field.
The term Free vortex, as used here, refers to a vortex flow where the fluid particles retain their orientation while the flow rotates around an axis (i.e., vorticity is zero) everywhere in the flow except near the central axis (where in mathematical terms, a singularity exists). Placing a hypothetical arrow moving with the fluid particles, the arrow continues to point in the same direction while it rotates around the axis with the flow. An ideal irrotational sink vortex could be an example of a free vortex.
The term sink vortex as used here refers to the actual flow field produced in the vicinity of a drainage region, said drainage could be by any means including natural drainage directed downward by gravity or drainage in any direction induced by pressure differential or other means.
The term Forced vortex, as used here, refers to a vortex flow where the fluid moves in a solid-body rotation; meaning that there is no shear in the flow and therefore the vorticity is constant everywhere and equal to 2ζ, where ζ is the rate of rotation. A hypothetical arrow pointing to the axis of rotation and attached to the fluid particles in a Forced vortex continues to point to the axis of rotation while rotating around the axis.
Cotyledon a part of a plant embryo (100) that becomes the embryonic first leaves of a seedling. The cotyledon is located at one end of a plant embryo opposite to the end where roots will eventually form (foot (102)). When there are several cotyledons, the may form a structure referred to as a crown (101).
Diameter of the crown refers to the diameter of a crown structure at its widest (103).
Length of a plant embryo refers to the linear distance from the tip of the root end to the tip of the cotyledon end measured along the longitudinal axis of the embryo (104).
The terms tube, channel and flow channel are used interchangeably. The terms are used without specific reference to any particular geometric shape of the cross-section, unless specifically stated otherwise.
The terms fluid dynamics and hydrodynamics are used interchangeably and refer to the same physical principles of flow of fluids.
Strain is the geometrical measure of deformation representing the relative displacement between points in the material body; it is represented as the ratio or percentage of deformation in relation to the original dimension.
Normal strain defines the ratio or percentage amount of stretch or compression along material line elements (ratio of the deformation to the original length in the direction of the deformation).
Shear strain defines the ratio or percentage amount of deformation relative to the original dimension associated with the sliding of material plane layers over each other.
Extensional strain is a normal strain where the element stretches.
Axially extensional strain is an element that stretches along the axial direction.
Radially extensional strain is an element that stretches along the radial direction.
Compressional strain is a normal strain where the element contracts.
Axially compressional strain refers to deformation of an element that contracts along the axial direction.
Radially compressional strain refers to deformation of an element that contracts along the radial direction.
Rate of Stain is the change in strain with respect to time
Hydraulic diameter, Dh, is a term used to characterize flow in noncircular tubes and channels. By definition, it is given by Dh,=4 A/S where A is the cross-sectional area of the noncircular tube or channel and S is the wetted perimeter of the cross-section.
Mean velocity in a channel is defined as the volumetric flow rate divided by the cross-sectional area of the channel.
Contraction ratio is defined as the ratio of the mean velocity at the outlet to the mean velocity at the inlet in a channel.
Mean stress is the stress that is averaged over a surface.
Mean rate of strain is the rate of strain averaged over a surface.
Dynamic viscosity of a fluid is the ratio of shear stress to rate of shear strain, a constant for a Newtonian fluid. Water, glycerin, silicone oil are examples of Newtonian fluids.
Rate of strain profile is a profile showing the variation of the rate of strain.
Unit of length in millimetre is abbreviated as “mm”.
Unit of rate of strain as reciprocal second is abbreviated as “1/s”.
In general, a flow with higher average rate of strain will impose higher average stress on a particle (or embryo) or on a cluster of particles (or cluster of embryos) suspended in the fluid.
The terms boundary layer, viscous boundary layer, and thin boundary layer are used interchangeably to mean the boundary layer formed by a forced rotating flow inside the circular container with or without the presence of a sink boundary layer.
TABLE 2List of designations pertaining to the Figures. 1House Frame 2Top support structure 3Bottom support structure 4Outer container 5Separator container 6Bottom wall of separator container 7Conduit of separator container 8Sensor 9Feed conduit 10Axial centre of separator container (5) 11Hollow shaft 12Hollow-shaft motor 13Upstream container 14Outlet of feed conduit (9) 15Liquid barrier of outer container 16Opening in the outer container 17Outer container draining tube 18Rotation means 19Base block 20Boundary layer 21Fluid level in the separator container at start 22Fluid level in the separator container during operation 25Secondary outlet 30Third outlet from container (5) 31Separator container (5) draining tube 32Extraction tube 33Bottom end of Extraction tube 34Hole in top support structure 35Linear actuator 36Valve, such as a pinch, gate, drop-through rotary or needlevalve, or a set of such valves (optional) 37Draining valve (optional) 38Controlling unit (optional) 50Inner diameter of a circular embodiment of an conduit (7) 51Inner diameter of a circular embodiment of a separatorcontainer (5) 52Inner diameter of an embodiment of rotating means 18 a 53Diameter of an embodiment of rotating means 18 b 54Height of separator container (5)200Bioreactor205System for processing somatic plant embryos210Extraction of embryogenic clusters215Transfer of embryogenic clusters220Disperser225Transfer of dispersed embryogenic mass230Separator233Transfer of Immature embryogenic tissue235Transfer of separated embryos240Dilutor245Transfer of Diluted embryos247Sorter reservoir249Test section250Detector-Sorter-Orienting System255Transfer of oriented embryos260Deposition of oriented mature embryos in embryo receivers263Fluid and rejected embryos265Transfer of Accepted mature embryos270Germination280Nursery290Dilution fluid300Plate with embryo containers305Perforations in the plate310Rejection reservoir312Embryo collector313Step motor/switch315Linear actuator of the x-/y-table320Substrate325Cavity in substrate330Open space between embryo containers335Connectors between embryo containers340Embryo container345Perforations in embryo container350Narrow hole360length of the free jet365Outlet370Straight section of tube before outlet 365375Flow direction of fluid and oriented embryos380Tube diameter381Encapsulating liquid382Encapsulating liquid delivery jet 383aOriented embryo inside the delivery jet 383bEmbryo inside an unstable delivery jet385Substantially stable delivery jet386Substantially unstable delivery jet387Vessel delivering the encapsulating liquid (tube)388Inner delivering tube401Segment of an axisymmetric channel402Segment of an axisymmetric channel403 to 440Dimensions according to Table 5441Connector tube481Segment of a non-axisymetric channel 481aCross section of 481482Segment of a non-axisymetric channel 482aCross section of 482442 to 490Dimensions according to Table 6501Fluid inlet502Inlet tube503Fluid outlet504Outlet tube505Reservoir tube506Reservoir device507Intersection508Inlet valve (optional)509Outlet valve (optional)510Orientation detector511Reservoir tube detector (optional)512Outlet tube detector (optional)518Flow direction519Three-way intersection valve (optional)521Secondary destination plate (optional)522Secondary outlet tube (optional)523Secondary intersection (optional)524Liquid drainage (optional)530Inlet/outlet openings of the intersection valve531Intersection valve house532Intersection valve rotor533Intersection valve rotor flow channel534Diameter of inlet/outlet540x, y-movable table device (optional)541Device for x, y-moving the outlet tube (504) (optional)550Three way valve at secondary intersection (523) (optional)560Tube air inlet/outlet to reservoir device (optional)561Air inlet/outlet (optional)562Air filter (optional)
TABLE 5List of dimension designations pertaining to FIG. 13.Cross sectionInner diameterPreferred Inner diameterposition[mm]for Norway Spruce(403) 3.0-10.0 9.0-9.5(404)2.0-9.0 5.0-5.5(405) 3.0-10.0 9.0-9.5(406)2.0-9.04.75-5.0(407) 3.0-10.0 9.0-9.5(408)2.0-9.0 4.0-4.25(409) 3.0-10.0 9.0-9.5(410)2.0-9.0 5.5-6.0(411)2.0-9.05.75-6.0(412)1.0-8.03.25-3.5(413)2.0-9.05.75-6.0(414)1.0-8.0 3.0-3.25(415)2.0-9.05.75-6.0(416)1.0-8.0 2.5-2.75(417)2.0-9.05.75-6.0(418)1.0-8.0 2.5-2.75(419)2.0-9.05.75-6.0(420)2.0-9.05.75-6.0Length on detailsLength [mm](421)30.0(422)10.0(423)30.0(424)5.0(425)30.0(426)5.0(427)20.0(428)10.0(429)20.0(430)30.0(431)5.0(432)30.0(433)5.0(434)30.0(435)5.0(436)30.0(437)5.0(438)20.0(439)10.0(440)10.0
TABLE 6List of dimension designations pertaining to FIG. 14Exemplified inner cross-section dimensionsInnerBlack arrow sideShape ofdimensions[mm]Width sideinner[mm](483)-(490)[mm]sectionAlt. 1Alt. 2Alt. 1Alt. 2Alt. 1Alt. 2(442)circular9.59.5(443)Rectangular(483) 5.0(483) 4.759.59.5(444)circular9.59.5(445)Rectangular(484) 9.5(484) 9.55.04.25(446)circular9.59.5(447)Rectangular(485) 5.0(485) 3.759.59.5(448)circular9.59.5(449)Rectangular(486) 9.5(486) 9.55.03.5(450)circular9.59.5(451)circular6.06.0(452)circular6.06.0(453)Rectangular(487) 3.5(487) 3.256.06.0(454)circular6.06.0(455)Rectangular(488) 6.0(488) 6.03.53.25(456)circular6.06.0(457)Rectangular(489) 3.5(489) 2.756.06.0(458)circular6.06.0(459)Rectangular(490) 6.0(490) 6.03.52.75(460)circular6.06.0